Since the launch of recombinant insulin nearly three decades ago, the protein therapeutics market has boomed to a multibillion industry totaling to $63 billion in 2007 worldwide ($39 billion in U.S. alone), and is projected to reach $87 billion in 2010. Amidst the great therapeutic potential of peptide or protein-based drugs, 96% of all drug candidates have been abandoned during preclinical or clinical development due to solubility or aggregation issues. Though it may proceed through different pathways, aggregation can possibly occur during any of the multiple stages of protein drug processing which may include fermentation, purification, formulation, and even storage—conditions that would subject the proteins under stress or in its non-native environment. Apart from potential decrease in efficacy, and reduced bioavailability, aggregation of protein therapeutics can be toxic leading to serious, detrimental effects such as renal failure and/or immunogenicity. In 2006, a catastrophic example was reported when healthy participants in a clinical trial for a monoclonal antibody drug aimed to boost T cells in the immune system suffered several severe side effects including vomiting, pain, and extreme swelling when the candidate drug was administrated. Despite prior testing showing no ill effects for mice and rats, the response of the human subjects to the synthetic antibody can still be life threatening.
One of the most used strategies in improving the pharmacokinetic property of protein drugs is by polyethylene glycol attachment (PEGylation). A highly flexible, and soluble polymer, PEG has been widely recognized and proven to be an acceptable chemical modification to reduce immunogenicity and aggregation in the protein therapeutics industry. Although PEGylation reduces protein aggregation by blocking undesired contacts with other proteins, large poly(ethylene glycol)s are used, m.w. between 2,000 to 30,000, which is of comparable size of the protein itself. Thus, the mechanism appears to be random large blocking instead of site selective blocking. This approach may suffer from the disadvantage of reducing the drug's specific activity by also blocking the active sites of the modified proteins. Furthermore, the size of the modified protein may vary due to molecular weight variation of the PEG.
Immunoglobulins (IgG) have been routinely isolated from mammalian sera for both diagnostic and therapeutic purposes. Polyclonal antibodies for example, are employed as ligands in immunoaffinity columns or as labeling agents to identify or quantify molecules in various assays such as enzyme-linked immunsorbent assay (ELISA), Western Blot, etc. Immunoglobulin therapy was first introduced in the early 1950's to treat patients who do not produce sufficient antibodies of their own by administering immunoglobulins intramuscularly. Protection from specific diseases or viruses has also been facilitated by hyperimmune IgG infusion. Due to the complex matrix of sera, and the heterogeneity of the size and charge of polyclonal IgG, conventional purification methods by precipitation with inorganic salts or alcohol, electrophoresis, gel permeation and immunoaffinity chromatography can be challenging for large scale IgG isolation.
Protein A from Staphylococcus aureus has been widely used in isolating IgG from mammalian sera. Due to its high affinity to the Fc domain of antibodies, Protein A can selectively isolate immunoglobulins from sera or any body fluids rapidly. Since Protein A isolation involves laborious and expensive procedures, supports immobilizing Protein A have been conventionally used for large scale protein purification to maximize the use of Protein A. Protein A immobilized on sepharose and sephacryl gels are now commercially available and are popularly used to isolate IgG's. However, leaching of protein A and hence contaminating the IgG isolates have been noted on sepharose gels. On the other hand, activation of sephacryl gels involves the use of toxic agents. A compromise between yield and purity has always been a problem with IgG isolation